MYL5 as a Novel Prognostic Marker is Associated with Immune Infiltrating in Breast Cancer: A Preliminary Study

Background Myosin light chain plays a vital regulatory function in a large-scale cellular physiological procedure, however, the role of myosin light chain 5 (MYL5) in breast cancer has not been reported. In this study, we aimed to elucidate the effects of MYL5 on clinical prognosis and immune cell infiltration, and further explore the potential mechanism in breast cancer patients. Methods In this study, we first explored the expression pattern and prognostic value of MYL5 in breast cancer across multiple databases, including Oncomine, TCGA, GTEx, GEPIA2, PrognoScan, and Kaplan–Meier Plotter. The correlations of MYL5 expression with immune cell infiltration and associational gene markers in breast cancer were analyzed by using the TIMER, TIMER2.0, and TISIDB databases. The enrichment and prognosis analysis of MYL5-related genes were implemented by using LinkOmics datasets. Results We found that there was a low expression of MYL5 in breast cancer than in corresponding normal tissue by analyzing the data from Oncomine and TCGA datasets. Furthermore, research showed the prognosis of the MYL5 high-expression group was better than the low-expression group in breast cancer patients. Furthermore, MYL5 expression is markedly related to the tumor-infiltrating immune cells (TIICs), including cancer-associated fibroblast, B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, and dendritic cell, and related to immune molecules as well as the associated gene markers of TIICs. Conclusion MYL5 can serve as a prognostic signature in breast cancer and is associated with immune infiltration. This study first offers a relatively comprehensive understanding of the oncogenic roles of MYL5 for breast cancer.


Introduction
Breast cancer, the most common malignancy in females, is a leading cause of cancer-related incidence and mortality around the world [1][2][3]. Breast invasive carcinoma (BRCA), the most common type of breast cancer, is generally poorly diferentiated and has a poorer prognosis than the other types, and is the most common type of breast cancer, accounting for approximately 80 percent of breast cancer [4]. With advances in early diagnosis and treatment, many patients have been successfully treated, with an average 5-year survival rate of about 90% [5]. However, about 20% to 25% of patients are diagnosed with locally advanced breast cancer, and early recurrence and death are the main causes of therapeutic failure in these patients [6]. Terefore, to further demonstrate the molecular mechanism of mammary carcinoma, it is pressing to probe new therapeutic methods.
Myosins consist of two heavy chains, two nonphosphorylable base light chains, and two phosphorylable regulatory light chains. Myosins have been shown to be involved in cell contraction, cell signaling, endocytosis, vesicle transport, and protein/RNA localization [7,8], and are the actin-dependent molecular motor that uses the energy hydrolyzed by ATP to move along actin flaments and generate force, which plays a key role in regulating tumor progression and metastasis [9][10][11]. Te changes in myosins expression could be used to predict therapeutic outcomes and, in some cases, provide attractive targets for the development of antitumor drugs [12]. Myosin light chain 5 (MYL5) encodes one of the light chains of myosin, which is a component of the hexameric ATPase cellular motor protein myosin. However, to date, there have been few clinical studies to indicate the clinical value and functional role of MYL5 in tumors, especially breast cancer. Terefore, in view of the complexity of tumor occurrence and progression in BRCA (breast invasive carcinoma), it is of great importance for us to clarify the correlation between MYL5 and clinical prognosis, as well as the potential molecular mechanism of great signifcance in breast cancer.
In this study, we attempted to explore the efect of MYL5 expression on the prognosis of patients with pan-cancer through bioinformatics analysis using public data sets, and further explore the potential molecular mechanism of MYL5 on the clinical prognosis of breast cancer.

Gene Expression Analysis.
We used the TIMER2.0 (Tumor Immune Estimation Resource, Version 2) and GEPIA2 (Gene Expression Profling Interactive Analysis, Version 2) tools to gain the MYL5 expression diference between pan-cancer and normal tissues. By using the GEPIA2, we analyzed the correlation between the expression of MYL5 and the pathological stages of tumors, and results were exhibited by using the box or violin fgure. Te data of these results in this study from the Oncomine database (https://www.oncomine.org) were obtained before its discontinuation.

Survival Prognosis Analysis.
We employed the "Survival Map" module of GEPIA2 to explore the efects of MYL5 expression on the OS (Overall survival) and DFS (Disease-free survival) across all cancers in the TCGA dataset. We also used the data from PrognoScan (https://dna00.bio. kyutech.ac.jp/PrognoScan/index.html) and Kaplan-Meier plotter (https://kmplot.com/analysis/) databases to further analyze the efects of the expression of MYL5 on outcome in cancers. Te efect of both MYL5 and clinicopathological factors on patient prognosis in breast tumor patients was analyzed by the Kaplan-Meier plotter tool.

Immune Infltrating Analysis and Prognosis Analysis.
We used the TIMER2 web server to gain the relationship between the expression of MYL5 and cancer-associated fbroblasts across all TCGA tumors. Te EPIC, MCPCOUNTER, XCELL, and TIDE algorithms were applied for immune infltration estimations. Te P values and partial correlation (cor) values were obtained via the purityadjusted Spearman's rank correlation test. Te data were displayed by a heatmap and a scatter plot. Additionally, the relationship between MYL5 expression and other immune infltration cells was determined by using the TIMER (https://cistrome.org/TIMER/) databases. Te relationship between the gene markers of TIICs and the expression of MYL5 were analyzed by GEPIA2 and TIMER2.0 tools.

TISIDB Database
Analysis. TISIDB database (https://cis. hku.hk/TISIDB) is a portal for tumor and immune system interactions that integrates multiple heterogeneous data types. We used the data from the TISIDB dataset to analyze the association between MYL5 expression and lymphocytes, immune modulators (including immunosuppressants and immunostimulants), and chemokines.

LinkedOmics Database Analysis.
We employed the LinkedOmics database (https://www.linkedomics.org/login. php) to explore 32 TCGA cancer-associated multidimensional databases. Te diferentially expressed genes correlated with MYL5 were screened from the TCGA BRCA queue by the LinkFinder module, and the association of Pearson correlation coefcient test results was displayed in the volcano map and heat map, respectively. Function module analysis of Gene Ontology biological process (GO_BP), Gene Ontology molecular function (GO_MF), Gene Ontology cellular component (GO_CC), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways by the Gene Set Enrichment Analysis (GSEA) in the link interpreter module.

Statistical Analysis.
Te data from the Oncomine database were presented as p values determined in t-tests, fold changes, and gene ranks. In their respective analyses, survival maps were generated using the PrognoScan, Kaplan-Meier Plotter, TIMER, TIMER2.0, and GEPIA2 databases, including HR and p values or p values from logrank tests. Spearman's and Pearson's correlation analyses were used to measure the degree of correlation between specifc variables. p < 0.05 was considered statistically signifcant, if not specially noted.

(b)
Expression -log 2 (TPM + 1) Expression -log 2 (TPM + 1)   Figure 1: Te expression levels of the MYL5 gene in pan-tumors and normal tissues. Te expression levels of MYL5 in diferent types of tumor tissues and normal tissues were analyzed by using the Oncomine database (the threshold of p value is 0.01, fold change is 2, and gene ranking is all) (a), TIMER 2.0 (b) and GEPIA2 (c) tools (d). Te expression levels of the MYL5 gene were analyzed by the main pathological stages of BRCA and TGCT. Log2 (TPM + 1) was applied for the log-scale. * p < 0.05, * * p < 0.01, and * * * p < 0.001.
( Figure 1(b)). Because there were no matched normal tissues for adrenocortical carcinoma (ACC), lymphoid neoplasm difuse large B-cell lymphoma (DLBC), acute myeloid leukemia (LAML), brain lower grade glioma (LGG), ovarian serous cystadenocarcinoma (OV), sarcoma (SARC), skin cutaneous melanoma (SKCM), testicular germ cell tumors (TGCG), thymoma (THYM), and uterine carcinosarcoma (UCS), we further investigated the diferential expression of MYL5 between tumors and normal tissues by using the GEPIA 2 tools to match TCGA normal and GTEx data. As shown in Figure 1(c), there was a higher expression of MYL5 in DLBC, LAML, and THYM than in corresponding control tissues, but a lower expression of MYL5 in SKCM and TGCT. Furthermore, we employed the GEPIA 2.0 tool to analyze the relationship between MYL5 expression and clinical stage, and the results showed that the expression of MYL5 in BRCA and TGCT correlated obviously with the clinical stage ( Figure 1(d)). Te data of this part indicated that MYL5 expression existed a signifcant diference between pan-cancer and normal tissue, which deserved further investigation.

Survival Analysis of MYL5 Expression by GEPIA 2 Tool.
To study the efect of the MYL5 gene on survival, we used the GEPIA 2 tool to analyze the data divided into highexpression and low-expression groups from TCGA and GEO databases, and the heatmap and Kaplan-Meier plot of overall survival (OS) and disease-free survival (DFS) were displayed (Figures 2(a) and 2(b)). We found that in BRCA patients, there was signifcantly longer overall survival (OS) in the high-expression of MYL5 group than in the lowexpression group (HR � 0.68, p (HR) � 0.018, and longrankp � 0.017), as well as in KIRC patients (HR � 0.62, p (HR) � 0.0021, and long-rankp � 0.0019). In addition, the disease free survival (DFS) of KIRC patients in the MYL5 high-expression group was longer than in the MYL5 low-expression group (HR � 0.43, p (HR) � 0.000011, and long-rankp � 0.0000057); however, the DFS of KICH (HR � 4.6, p (HR) � 0.055, and long-rankp � 0.035) and UVM (HR � 3.3, p (HR) � 0.02, and long-rankp � 0.013) patients in MYL5 high-expression group was lower than in MYL5 low-expression group. All data manifested that the MYL5 gene could be a potential and novel prognosis factor and it could be of beneft to clinical diagnosis and therapy for diferent cancers.  3(l), displayed that the OS and PPS in the MYL5 high-expression group were shorter than the MYL5 low-expression group. All data from the Kaplan-Meier plotter dataset of this part demonstrated that MYL5 could be a potential and poor prognostic factor for lung cancer and gastric cancer patients, but a better prognostic biomarker for breast cancer and ovarian cancer patients. To further verify the conclusion among the previous data about the efect of the MYL5 gene on prognosis, we used the data from the PrognoScan dataset to study whether MYL5 expression had contributed to a better prognosis for special types of cancers. As shown in Figures 3(m) and 3(n), the RFS and DMFS of breast cancer patients in the MYL5 high-expression group were signifcantly longer than the MYL5 low-expression group, which kept in with the survival data from the Kaplan-Meier plotter dataset and these data further indicated that MYL5 served as a potential and favorable biomarker on diagnosis in breast cancer. Similarly, as for lung cancer, the result of OS in the Prog-noScan dataset supported the conclusion that the OS of lung cancer patients in the MYL5 high-expression expression group was shorter than the MYL5 low-expression group, indicating that MYL5 could be a poor prognostic factor for lung cancer (Figure 3(o)). However, in the ovarian cancer survival data from the PrognoScan dataset (n � 1656), we found that the OS in MYL5 high-expression group was shorter than the MYL5 low-expression group, which contained to the analysis of survival data from the Kaplan-Meier plotter dataset (n � 123) (Figure 3(p)), and we speculated that the contradictory results could be caused by the number of samples. In addition, we also found the prognostic difference between the MYL5 high-expression group and the low-expression group among colorectal cancer, soft tissue cancer, acute myelocytic leukemia (AML), and multiple myeloma (MM  CHOL  COAD  DLBC  ESCA  GBM  HNSC  KICH  KIRC  KIRP  LAML  LGG  LIHC  LUAD  LUSC  MESO  OV  PAAD  PCPG  PRAD  READ  SARC  SKCM  STAD  TGCT  THCA  THYM  UCEC  UCS

Correlation Analysis between MYL5 Expression and
Cancer-Associated Fibroblast Infltration. After conducting a prognosis analysis, we frst further investigated the relationship of MYL5 expression with cancer-associated fbroblasts (CAF). Te correlation of MYL5 expression with CAF infltrate in pan-cancer was exhibited by the heatmap through employing the TIMER2.0 tool, with the EPIC, MCPCOUNTER, XCELL, and TIDE algorithms, respectively ( Figure 4(a)). Ten, the scatter diagrams showed the detailed contact between the two. In BRCA-LumB, HNSC, and LUAD, the MYL5 expression was signifcantly and negatively correlated to the CAF infltrate by using EPIC, MCPCOUNTER, and TIDE algorithms, but was positively related to the CAF infltrate via XCELL algorithm (Figures 4(b), 4(d), and 4(e)). Interestingly, as shown in Figure 4(c), we found that in COAD, the MYL expression obviously and negatively correlated with the CAF infltrate through the four algorithms; however, in TGCT, the expression of MYL5 markedly and positively related to the infltrate of CAF via the four algorithms (Figure 4(f )).

Prognostic Analysis of MYL5 Expression and Immune Infltrate Cells in Diferent Molecular Subtypes of BRCA.
In this part, we studied the efect of MYL5 expression and immune infltrate cells, including B cell, CD8 + T cell, CD4 + T cell, macrophage, neutrophil, and dendritic cell (DC) on prognosis in BRCA, BRCA-Basal, BRCA-Her2, and BCRA-Luminal, respectively. In BRCA patients who were not divided into diferent molecular subtypes, we found that MYL5 expression and B cell infltrate collectively afected the  (Figure 6(a)). If distinguishing molecular subtypes in BRCA, we detected that in BRCA-Basal, all indexes did not exert an impact on the patient's prognosis; however, in BRCA-Luminal, lowexpression of MYL5 indicated a poor prognosis, and in BRCA-Her2, the high-level infltrate of B cell displayed a great prognosis (Figures 6(b)-6(d)). Te analysis of these data via employing the TIMER tool suggested that MYL5 might be great prognostic biomarkers for BRCA-Luminal patients.

Correlation Analysis of MYL5 with Immnue Cell Marks in BRCA.
To further investigate the potential relationship between MYL5 expression and TIICs in BRCA, we explored the correlations between MYL5 and several immune cell markers, including B cell, M1/M2 macrophage, tumor-associated macrophage (TAM), monocyte, and T cell exhaustion, by TIMER and GEPIA2. As shown in Figure 7(a), MYL5 expression signifcantly correlated with B cell markers, inculding KRT20 (CD20), CD19, and CD38. As for M1 macrophage markers, NOS2 and PTGS2 (COX) also obviously correlated with the expression of MYL5 (Figure 7(b)). CD163, MS4A4A, and VSIG4 served as the associational markers of M2 macrophage and were correlated with MYL5 expression (Figure 7(c)). Te gene markers of TAM, including CCL2, CD68, and IL-10, are markedly related to the expression of the MYL5 gene in BRCA (Figure 7(d)). We found that CD86 and CD16 (FCGR3A) as gene markers for monocyte were signifcantly and negatively relevant to MYL5 expression (Figure 7(e)). Especially, the associational markers of T cell exhaustion including PD1, PD-L1, and CTLA4 were deemed to correlate with tumor immune escape, and the results exhibited that the expression of MYL5 negatively and signifcantly correlated with them, which additionally indicated that MYL5 could serve as a good prognostic biomarker for BRCA patients. To verify the conclusion about the association between the expression of MYL5 and immune cell markers, we employed the GEPIA2 tool to analyze the relationship of MYL5 with B cell, M1/M2 macrophage, TAM, monocyte, and T cell exhaustion in breast cancer and normal tissue, severally. As shown in Table 3, the results of the correlation between MYL5 expression and gene markers of B cell, M1/M2 macrophage, TAM, monocyte, and T cell exhaustion brought into correspondence with the previous result in Figure 7, showing that these markers all negatively and signifcantly correlated with MYL5 expression in BRCA. Particularly, we found that NOS2, CCL2, CD68, CD86, CD16 (FCGR3A), and PD-L1 (CD274) also have a signifcant correlation with the expression of MYL5. Te data of this part demonstrated that MYL5, a novel, and prognostic biomarker, signally correlated with the immune cell infltration and their correlative gene markers.

Relation between MYL5 with Immune Molecules.
To further improve the cognition of the relationship between MYL5 expression and immune infltration, we studied the associations between the expression of the MYL5 gene and        (Figure 8(e)). Similarly, in Figure 8(f ), the association between MYL5 expression and receptors in pan-cancer was also presented by heatmap, and the top 6 scatter plots of the absolute p values showed the correlation of the two in BRCA. Terefore, it was confrmed that MYL5 participated widely in modulating various immune molecules in BRCA to afect immune infltration in the tumor microenvironment.

Coexpression Network of MYL5 Gene in Breast Cancer.
To further investigate the biological function of MYL5 in breast cancer, we used the data from the LinkedOmics dataset to explore the coexpression pattern of MYL5 in TCGA-BRCA. As shown in Figure 9(a), it displayed that 7785 genes (dark red dots) positively correlated with MYL5, and 7613 genes (dark green dots) negatively correlated with MYL5 (Figures 9(b) and 9(c)). What is noteworthy is that as shown in Figure 9(d), the top 48 (two genes (LOC284441 and KIAA0754) were not found in GEPIA2) negatively genes with a highly owned probability of becoming high-risk markers in BRCA, of which 36/48 genes had a disadvantageous hazard ratio (HR), especially CDK8 and NUP205. In contrast, there were 33 of the top 50 genes with protective HR in the top 50 positively signifcant genes, mainly including CCDC24, UBXN11, PCP2, and TNFRSF14 (Figure 9(e)). Te results of GO analysis (Biological Process) showed that coexpressed genes of MYL5 mainly participated in mitochondrial respiratory chain complex assembly, NADH dehydrogenase complex assembly, chromosome segregation, cell cycle G1/S phase transition, etc. (Figure 9(f )). As shown in Figure 9(g), GO analysis (molecular function) displayed that MYL5 mainly joined structural constituent of ribosome, helicase activity, ATPase activity, histone binding, etc. Coexpressed genes of MYL5 primarily took part in the respiratory chain, NADH dehydrogenase complex, mitochondrial membrane part, chromosomal, etc. via GO analysis (cellular component) (Figure 9(h)). Te bar chart of KEGG pathways analysis from the LinkedOmics database  revealed that coexpression genes of MYL5 are mainly involved in ribosome, oxidative phosphorylation, arachidonic acid metabolism, cell cycle, etc. mRNA surveillance pathway might be mainly involved in the efect of coexpression genes of MYL5 on breast cancer pathogenesis (Figure 9(i)).
Te data of this part indicated a wide infuence of the MYL5 expression network on the prognosis, proliferation, and metabolism of BRCA.

Discussion
Recent studies showed that myosins play vital roles in the physiological or pathological processes of cells, which included cytokinesis failure, chromosomal [13] and centrosomal amplifcation [14], multipolar spindle formation, and DNA microsatellite instability [10]. Tese progresses were all preconditions of cancer formation and development. Furthermore, myosins activated many processes of malignancy invasion and metastasis, mainly including cell migration, adhesion, protrusion formation, cycle arrest, and apoptosis inhibition [15]. Recently, studies have incrementally indicated that the myosin superfamily played an important role during oncogenesis and tumor-related diseases [16][17][18]. For example, Myosin light chain (MLC) kinase inhibitors could block the invasion and adhesion of human pancreatic tumor cell lines [19]. Te upregulation of myosin VA by Snail was involved in tumor cell migration and metastasis [20]. As the research progresses, the presence of myosin II, such as MYL1, MYL2, and MYL9 in the nuclei of several cell types, and their transcriptional function is gradually reported [21,22]. However, little was known about the existence and tumorigenic role of MYL5 in many types of tumors. In a previous study, we used the expression of WDR6 (WD repeat domain 6) between pan-cancer and normal tissue by employing the bioinformation analysis and exploring the immunological and prognostic role in lung cancer patients [23]. Similarly, in this study, we frst analyzed the expression of the MYL5 gene between pancancer and normal tissues, fnding that by analyzing the data of the Oncomine database, compared with corresponding normal tissues, MYL5 were underexpressed in breast cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, and leukemia, but were highly expressed in kidney cancer. Meanwhile, our results from the TCGA database showed that the expression of the MYL5 gene was signifcantly elevated in KIRC, LIHC, and PRAD, compared to the expression of the MYL5 gene in normal tissues, but was markedly decreased in BRCA, COAD, HNSC, KICH, LUAD, and THCA. Terefore, the expression diference of MYL5 between cancer tissues and normal tissues was worth further investigation, in order to reveal the value of MYL5 in clinical diagnosis and therapy of tumor patients.
Myosins played a vital function not only in tumorigenesis but also could be used as an impressible signature for cancer diagnosis [24]. Studies showed that MYO5B (myosin VB) may become an important biomarker for gastric cancer because the expression of MYO5B was downregulated in gastric cancer and the inactivation of MYO5B may contribute to tumorigenesis [25]. Myosin VI (MYO6), serving as a sensitive biomarker, was highly expressed in the Golgi apparatus of prostate cancer cells [26]. In the present study, in order to elucidate the potential value of MYL5 on breast cancer, we investigated the efects of MYL5 expression on survival prognosis, mainly including OS, RFS, DMFS, PFS, FP, and PPS. Te results showed that in Kaplan-Meier       THCA  TGCT  STAD  SKCM  SARC  READ  PRAD  PCPG  PAAD  OV  MESO  LUSC  LUAD  LIHC  LGG  KIRP  KIRC  KICH  HNSC  GBM  ESCA  COAD  CHOL  CESC  BRCA THCA  TGCT  STAD  SKCM  SARC  READ  PRAD  PCPG  PAAD  OV  MESO  LUSC  LUAD  LIHC  LGG  KIRP  KIRC  KICH  HNSC  GBM  ESCA  COAD  CHOL  CESC  BRCA  BLCA  ACC   -0   1   -1   ULBP1   IL2RA   HHLA2   CD40   C10orf54  CD27  CD276  CD28   CD40LG  CD48  CD70  CD80  CD86  CXCL12  CXCR4  ENTPD1   ICOS  ICOSLG   IL6  IL6R  KLRC1  KLRK1  LTA  MICB  NT5E  PVR  RAET1E  TMEM173  TMIGD2  TNFRSF13B  TNFRSF13C  TNFRSF14  TNFRSF17  TNFRSF18  TNFRS25  TNFRSF4  TNFRSF8  TNFRSF9  TNFSF13  TNFSF13B  TNFSF14  TNFSF15  TNFSF18  TNFSF4  TNFSF9 (c)  UCEC  THCA  TGCT  STAD  SKCM  SARC  READ  PRAD  PCPG  PAAD  OV  MESO  LUSC  LUAD  LIHC  LGG  KIRP  KIRC  KICH  HNSC  GBM  ESCA  COAD  CHOL  CESC  BRCA  BLCA CCDC159  PCSK4  PCP2  HEXDC  NME3  FBXW4  ZBTB48  D2HGDH  CPLX1  CHCHD5  FAM128B  HEMK1  JMJD7-PLA2G4B  COQ4  TMEM115  ANKS3  APBB3  TBC1D17  SH3GLB2  TCEA2  TNFRSF14  C7orf59  LRRC56  SNRNP70  C5orf45  SIGIRR  TNIP2  TEX264  ZNF580  PHPT1  RGS 12 Z-Score >3 1 0 -1 <-3  plotter and PrognoScan databases, the OS, DMFS, RFS, and PPS of breast cancer patients were signifcantly prolonged in the MYL5 high-expression group, compared with the MYL5 low-expression group. However, we found that in lung cancer, the OS, FP, and PPS in MYL5 high-expression group were markedly shorter than the MYL5 low-expression group. Te OS, progression-free survival (PFS), and PPS in the MYL5 high-expression group for ovarian cancer patients were obviously longer than in the MYL5 low-expression group. As for gastric cancer patients, the OS and PPS in the MYL5 high-expression group were shorter than the MYL5 low-expression group. All data from the Kaplan-Meier plotter dataset of this part demonstrated that MYL5 could be a potential and poor prognostic factor for lung cancer and gastric cancer patients, but a better prognostic biomarker for breast cancer and ovarian cancer patients. To further verify the conclusion among the previous data about the efect of the MYL5 gene on prognosis, we used the data from the PrognoScan dataset to study whether MYL5 expression had contributed to a better prognosis for special types of cancers. All data analysis from the PrognoScan database showed results that were mainly consistent with the Kaplan-Meier database, except for ovarian cancer. We speculated that the contradictory survival analysis results between the two databases for ovarian cancer could be mainly caused by the number of samples. Tis was an interesting phenomenon, which was worthy of further molecular experiments and animal models to verify its accuracy in the future. Terefore, all data of this part demonstrated that MYL5 might be a potential and novel biomarker for specifc cancer patients' clinical diagnosis and therapy.

Te Breast Journal
Tumor cells exist in a complex tumor microenvironment (TME) [27]. It is all known that tumorinfltrating immune cells, as prominent components of the tumor microenvironment, were closely linked to the initiation, progression, or metastasis of neoplasm [28,29]. In most cases, the primary role of TME exerted immunosuppression, which blocked anticancer immunity and sustain tumor progression [30]. Te immunosuppressive efect of TME was regulated by all immune cell types with immunomodulatory activities [27]. Studies showed that macrophages situated in TME tend to become tumor-associated macrophages (TAMs) to drive tumor progression, invasion, and metastasis [31]. In our study, we found that with the analysis data via using TIMER and GEPIA tool, MYL5 expression negatively correlated with macrophages infltration in breast tumor patients, and negatively and markedly correlated with the gene markers of macrophages and TAMs, indicating that MYL5 might afect the prognosis via regulating the TAMs in TME, thereby causing in longer survival in breast tumor patients. In addition, deeper and more research indicated that tumor-infltrating dendritic cells (DC) are inclined to promote immunosuppression and tolerance in TME, rather than drive anticancer immunity [32]; Our results showed that the expression of the MYL5 gene was negatively relevant to DC infltration in breast cancer, and this could suggest that MYL5 gene could afect the prognosis by regulating DC infltration level. Neutrophils which were recruited into cancer, are usually polarized towards the N2-subtype with protumor roles [32]. Our study found that MYL5 expression also correlated with neutrophils. T cells are the main expression cells of anticancer response. Most tumors reduced T cell-mediated immune response in various ways, including inhibiting T cell transport to the tumor, interfering with antigen-presenting cells, and efector T cells [30,33]. Tumor cells expressed PD-L1 (programmed cell death protein ligand 1) or PD-L2 (programmed cell death protein ligand 2) ligands that match the T-cell PD-1 protein, preventing them from fnding the tumor and sending signals to the immune system to attack the tumor, directly leading to T-cell failure [34]. Clinically, blocking this event, namely the application of anti-PD-1 and anti-PD-L1 antibodies, could not only promote the proliferation of T cells but also restore the cytotoxic response of T cells to tumor cells [30,34]. In the present study, MYL5 expression negatively correlated with the markers of T cells exhaustion, such as PDCD1 (PD-1), CD274 (PD-L1), and CTLA4 (cytotoxic Tlymphocyte antigen 4), which indicated that MYL5 could play a positive role in prolonging survival prognosis for breast cancer patients. Te LinkedOmics database analysis further showed that MYL5 not only has an important infuence on the prognosis of BRCA patients but also most of the genes coexpressed with MYL5 in BRCA are positively or negatively correlated with the prognosis of BRCA patients. In addition, these coexpressed genes were signifcantly focused on ATP-related and metabolism-related pathways, which coincided with the known function of MYL5. All signs indicate that MYL5 can play a vital role in afecting the prognosis of BRCA.
Our previous data manifested that the low-expression level of MYL5 was associated with poor prognosis, and further suggested that MYL5 could serve as a good prognostic biomarker to diagnose and treat breast cancer. Followingly, we further investigated the correlation between MYL5 expression and immuneinfltrating cells in the tumor microenvironment, and results showed that MYL5 expression signifcantly correlated with immune-infltrating cells and their gene markers in breast cancer patients. Furthermore, we integrated the information on MYL9-binding components and MYL9 expression-related genes in breast cancer for a series of enrichment analyses. However, we just used public databases of Oncomine, Kaplan-Meier plotter, GEO, PrognoScan, TCGA, TISIDB, and LinkedOmics datasets to demonstrate the efects of MYL5 on prognosis and immune infltration in breast tumors. Additionally, we still need more evidence from cell and animal levels in detail.

Conclusion
In brief, we concluded that there is a possible prognostic molecular marker for good survival correlated with immune cell infltration in breast cancer, called MYL5 expression. Te low-expression level of MYL5 leads to the worsening of clinical features (primary tumor scope, lymphatic metastasis, and pathological stage of tumor and prognosis). Tis study frstly ofers a relatively comprehensive understanding of the oncogenic roles of MYL5 for breast cancer. Relapse-free survival PPS: Postprogression survival PFS: Progression-free survival TME: Tumor microenvironment TAMs: Tumor-associated macrophages.

Conflicts of Interest
Te author declares that there are no conficts of interest.
Te Breast Journal